The mechanism proposed for the catalytic action of the pancreatic enzyme carboxypeptidase A on esters and peptides involves the postulated intermediacy of anhydrides formed between the carbonyl group of the substrate and the carboxylate group of Glu-270. By working at sub-zero temperatures in mixed aqueous-organic solvents using the ester substrate O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate, we were able to detect spectrophotometrically for the first time an acyl-carboxypeptidase (anhydride) intermediate. With the low temperature methodology for the detection of acyl-carboxypeptidases in hand, we are now concentrating heavily on studies of the roles of metal ions present at the active sites of various metallocarboxypeptidase A species such as the Co(II), Ni(II), Mn(II), Cd(II) and Hg(II) forms. This research is being performed by examining the kinetics of hydrolysis of selected substrates over a wide temperature range, employing both the modified and unmodified enzymes. In addition, comparative studies have been undertaken with the related pancreatic enzyme, carboxypeptidase B. Finally, we have discovered that carboxypeptidase A catalyzes the enolization of the ketonic substrate analog (minus)-3-p-methoxybenzoyl-2-benzylpropionic acid in a stereospecific manner. The study of this enolization reaction using the various metallocarboxypeptidases should allow us to examine the catalytic properties of the enzymebound nucleophilic groups and the active site metal ion without the complications of the formation and breakdown of a multiplicity of reaction intermediates. The results obtained should complement those found in our examination of hydrolysis reactions.